Lipopolysaccharide (LPS) is the main outer membrane surface-associated component in Gram-negative bacteria and is associated with an array of pathological and physiological activities in mammalian host immune responses. LPS-mediated toxicity caused by these bacteria is generally due to lipid A, the hydrophobic moiety of LPS, which can function as an agonist of Toll-like receptor 4 (TLR4)/MD-2. Lipid A comprises two bisphosphorylated glucosamine residues with six acyl chains attached.
Kdo (3-deoxy-DD-manno-octulosonate) is considered an essential component of LPS, and it is believed that the minimal LPS structure required for growth of E. coli is two Kdo residues attached to lipid A (Kdo2-lipid A; Re-LPS). Biosynthesis of Kdo begins with API (DD-arabinose 5-phosphate isomerase), which coverts DD-ribulose 5-phosphate (Ru5P) into A5P (D-arabinose 5-phosphate). In E. coli K-12, there are two API genes, kdsD and gutQ. Next, A5P is condensed with phosphoenolpyruvate (PEP) to form Kdo 8-phosphate (Kdo8P), which is then hydrolyzed forming Kdo. Kdo is subsequently activated as the sugar nucleotide CMP-Kdo, which is ultimately transferred to lipid IVA forming Kdo2-lipid IVA. The Kdo-dependent acyltransferases LpxL and LpxM transfer laurate and myristate, respectively, to Kdo2-lipid IVA to form Kdo2-lipid A (Re-LPS).
The strain E. coli K-12 TCM15, which has both API genes (kdsD and gutQ) deleted and thus lacks Kdo, is not viable unless supplied with exogenous A5P (e.g., see Meredith and Woodard, Identification of GutQ from Escherichia coli as a D-arabinose 5-phosphate isomerase, J Bacteriol 187:6936, 2005). The present invention features viable Gram-negative bacteria that substantially lack a ligand that acts as an agonist of TLR4/MD-2. Additional information regarding TLR4/MD-2 may be found at, for example, Qureshi et al., J. Exp. Med., Vol. 189, No. 4, Feb. 15, 1999, Pages 615-625; Shimazu et al., J. Exp. Med., Volume 189, Number 11, Jun. 7, 1999, Pages 1777-1782; Poltorak et al., Science 282, 2085 (1998), the disclosures of which are incorporated in their entirety by reference herein to the extent that the disclosures are consistent with the present invention. The ligand, for example, may comprise lipid A or a 6-acyl lipid. The viable Gram-negative bacteria comprise suppressor mutations, which enable viability despite lacking otherwise essential Kdo. Mamat et al. have described suppressor mutations in the yhjD and msbA genes non-conditional of E. coli TCM15 derivatives (see Mol Microbiol 67(3):633, 2008).
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.